define active site using a similar enzyme (active site is probably the Mg ions - but align another structure
Target Discovery: http://www.jbc.org/content/286/51/44197.long *Target (protein/gene name): STP1, S. agalactiae serine/threonine phosphatase *NCBI Gene # or RefSeq#: NCBI Gene ID3686217 *Protein ID (NP or XP #) or Wolbachia#:YP_329040 (used Caroline's Oligo Design - Dr. B090313) Get your NCBI NP# :YP_329040 **http://www.ncbi.nlm.nih.gov/protein/YP_329040.1** PDB #: 2PK0/ UNITPROTKB: Q3K363 *Organism: Streptococcus agalactiae A909 Etiologic Risk Group: *Background/Disease Information: The disease is called neonatal septicemia or neonatal sepsis. It is a blood infection that occurs in infants younger than 90 days old.
A number of different bacteria, including Escherichia coli (E.coli), Listeria, and certain strains of streptococcus, may cause neonatal sepsis.
Early-onset neonatal sepsis most often appears within 24 hours of birth. The baby gets the infection from the mother before or during delivery. Essentiality of this protein: In Group B Streptococcus (GBS), Stp1 controls serine/threonine kinase function, post-transcriptional regulation of
hemolysin, autolysis, and virulence. Although not essential for growth, Stp1 is critical for GBS pathogenesis. Complex of enzymes: No *EC#: 3.1.3.16
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): reagents are phosphoprotein and water. It is spectrophotometric. Not a couple assay. -- link to Sigma (or other company) page for assay or assay reagents (substrates) http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US -- link to paper that contains assay information http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=666937 -- List cost and quantity of substrate reagents and supplier: Reagent is pNPP/ phosphoprotein. Cost on SIGMA $71.00. SKU-Packsize is P7998-100mL. Structure Available (PDB or Homology model)
Here's the PDB crystal structure of a yeast low molecular weight protein tyrosine phosphate complexed with the substrate PNPP.
Here's the PDB structure for the target protein, streptococcus agalactiae:
from the Binding DB shows only 11 different molecules for my target. Expression Information (has it been expressed in bacterial cells): Escherichia coli bl21(de3) Purification Method: single-wavelength anomalous dispersion phasing and molecular replacement Image of protein (PyMol or etc):
*Length of protein in Amino Acids: 245 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 26732.7 Molar Extinction coefficient of your protein at 280 nm wavelength: 18450
*GC% Content for gene (codon optimized):
52.30352303523% Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): Link to DNA Works output
Primer design results for 'tail' primers (this is just 2 sequences): 100112 - These aren't tail primers -- Dr. B Upstream Primer: 5’ TACTTCCAATCCATGGAAATCTCTCTCCTG 3’ (<-- Whose information is this???) (Caroline)
GC Content: 43%
0 mM Mg2+ Tm:59.7˚C 1.5 mM Mg2+ Tm: 66.9˚C 2mM Mg2+ Tm: 67.4˚C
Table 1: GC content and melting temperature as a function of the concentration of Mg++ for CORRECTED** forward and reverse primers for insertion of S. agalactiae serine/threonine phosphatase gene into pNIC-Bsa4 vector with 0.4 uM Oligo concentration, 50 mM Na+ concentration, 0.3 mM dNTP concentration
Target Discovery:
http://www.jbc.org/content/286/51/44197.long
*Target (protein/gene name): STP1, S. agalactiae serine/threonine phosphatase
*NCBI Gene # or RefSeq#: NCBI Gene ID3686217
*Protein ID (NP or XP #) or Wolbachia#:YP_329040 (used Caroline's Oligo Design - Dr. B090313)
Get your NCBI NP# :YP_329040
**http://www.ncbi.nlm.nih.gov/protein/YP_329040.1**
PDB #: 2PK0/ UNITPROTKB: Q3K363
*Organism: Streptococcus agalactiae A909
Etiologic Risk Group:
*Background/Disease Information: The disease is called neonatal septicemia or neonatal sepsis. It is a blood infection that occurs in infants younger than 90 days old.
A number of different bacteria, including Escherichia coli (E.coli), Listeria, and certain strains of streptococcus, may cause neonatal sepsis.
Early-onset neonatal sepsis most often appears within 24 hours of birth. The baby gets the infection from the mother before or during delivery.
Essentiality of this protein: In Group B Streptococcus (GBS), Stp1 controls serine/threonine kinase function, post-transcriptional regulation of
hemolysin, autolysis, and virulence. Although not essential for growth, Stp1 is critical for GBS pathogenesis.
Complex of enzymes: No
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.16
Screenshot of BRENDA enzyme mechanism schematic:
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): reagents are phosphoprotein and water. It is spectrophotometric. Not a couple assay.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US
-- link to paper that contains assay information
http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=666937
-- List cost and quantity of substrate reagents and supplier: Reagent is pNPP/ phosphoprotein. Cost on SIGMA $71.00. SKU-Packsize is P7998-100mL.
Structure Available (PDB or Homology model)
Here's the PDB crystal structure of a yeast low molecular weight protein tyrosine phosphate complexed with the substrate PNPP.
Here's the PDB structure for the target protein, streptococcus agalactiae:
-- PDB # or closest PDB entry if using homology model: 2PKO - direct number but not for the A909 strand
-- Query Coverage (if not direct match): 98%
-- Max Ident (if not direct match): 495
Druggable Target (see Databases for this): Yes
Current Inhibitors:
http://www.bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?target=Serine%2Fthreonine-protein+phosphatase&tag=tg&kiunit=nM&icunit=nM&column=ki&submit=Search&energyterm=kJ%2Fmole
from the Binding DB shows only 11 different molecules for my target.
Expression Information (has it been expressed in bacterial cells): Escherichia coli bl21(de3)
Purification Method: single-wavelength anomalous dispersion phasing and molecular replacement
Image of protein (PyMol or etc):
*Amino Acid Sequence:
MEISLLTDIGQRRSNNQDFINQFENKAGVPLIILADGMGGHRAGNIASEMTVTDLGSDWA
ETDFSELSEIRDWMLVSIETENRKIYELGQSDDYKGMGTTIEAVAIVGDNIIFAHVGDSR
IGIVRQGEYHLLTSDHSLVNELVKAGQLTEEEAASHPQKNIITQSIGQANPVEPDLGVHL
LEEGDYLVVNSDGLTNMLSNADIATVLTQEKTLDDKNQDLITLANHRGGLDNITVALVYV
ESEAV.
*Length of protein in Amino Acids: 245
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 26732.7
Molar Extinction coefficient of your protein at 280 nm wavelength: 18450
*CDS Gene Sequence:
>gi|76786714:337442-338179 Streptococcus agalactiae A909 chromosome, complete genome ATGGAAATATCTCTTTTAACAGATATTGGGCAACGTCGTTCCAATAATCAAGATTTTATTAATCAATTTG AAAATAAAGCCGGCGTCCCACTAATCATCTTAGCAGATGGTATGGGAGGACATCGTGCAGGAAATATTGC TAGCGAGATGACTGTAACTGATCTAGGAAGTGATTGGGCGGAGACAGATTTTTCTGAGTTATCAGAAATT CGTGATTGGATGCTTGTCTCTATTGAAACAGAGAATCGCAAAATATATGAATTAGGTCAGTCAGATGATT ACAAAGGTATGGGAACTACCATTGAAGCAGTTGCCATTGTAGGAGATAATATTATTTTCGCACATGTAGG AGATTCACGTATCGGTATCGTTCGTCAAGGAGAGTATCATTTATTGACGAGTGATCATTCTCTTGTTAAT GAATTGGTAAAAGCTGGCCAATTAACAGAAGAAGAAGCTGCTAGTCATCCTCAAAAAAATATTATTACGC AATCAATTGGTCAAGCCAATCCAGTCGAACCTGATTTAGGAGTTCATCTACTTGAAGAAGGTGACTACCT TGTAGTTAATAGTGATGGACTAACTAATATGTTGAGTAATGCCGATATTGCTACAGTACTAACACAAGAA AAAACCTTAGATGATAAGAATCAAGATTTAATCACTCTAGCAAACCATAGAGGAGGTCTTGATAATATCA CTGTGGCACTAGTTTACGTTGAAAGTGAGGCGGTTTAA
*GC% Content for gene: 37.127371273713 %
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:
1 ATGGAAATCTCTCTCCTGACTGACATCGGTCAACGTCGCTCTAATAACCAGGACTTCATC
61 AATCAGTTCGAAAACAAGGCCGGTGTTCCGCTCATCATCCTGGCGGACGGCATGGGCGGT
121 CACCGTGCGGGTAACATTGCGAGCGAAATGACCGTTACCGATCTGGGCTCTGACTGGGCG
181 GAAACCGACTTCTCTGAACTGTCTGAAATCCGTGACTGGATGCTCGTTTCTATCGAAACG
241 GAAAACCGTAAAATCTACGAACTGGGTCAGTCTGACGACTACAAAGGTATGGGTACCACC
301 ATCGAAGCGGTTGCGATCGTTGGCGACAACATCATCTTCGCGCACGTTGGTGACTCTCGT
361 ATCGGTATCGTTCGTCAGGGTGAATACCATCTGCTGACTTCCGACCACTCTCTGGTTAAC
421 GAGCTGGTGAAAGCGGGTCAACTGACCGAAGAAGAAGCGGCGTCTCACCCGCAGAAGAAT
481 ATCATCACCCAGTCTATTGGCCAGGCGAACCCGGTTGAACCGGACCTGGGCGTCCACCTG
541 CTGGAAGAAGGTGACTACCTGGTTGTTAACTCTGACGGTCTGACCAACATGCTGTCTAAC
601 GCGGACATCGCGACCGTTCTGACGCAGGAAAAAACCCTGGACGACAAAAATCAGGACCTG
661 ATCACTCTCGCTAACCATCGTGGTGGTCTGGACAATATTACCGTTGCGCTGGTATACGTT
721 GAATCTGAAGCTGTTTAA
*GC% Content for gene (codon optimized):
52.30352303523%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
Link to DNA Works output
Tail Primer Designs: Entered 09/15/13 by Brandy
Brandy: https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-VUNVTnM0UC1SYVU
Caroline: https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-Wk9IcXdqWmItRHM
Primer design results for 'tail' primers (this is just 2 sequences): 100112 - These aren't tail primers -- Dr. B
Upstream Primer: 5’ TACTTCCAATCCATGGAAATCTCTCTCCTG 3’ (<-- Whose information is this???) (Caroline)
GC Content: 43%
0 mM Mg2+ Tm:59.7˚C 1.5 mM Mg2+ Tm: 66.9˚C 2mM Mg2+ Tm: 67.4˚C
4mM Mg2+ Tm: 68.5˚C 6mM Mg2+ Tm: 69.0˚C
Downstream Primer: 5’ GAATCTGAAGCTGTTTAACAGTAAAGGTGGATA 3’
GC Content: 36.4%
0 mM Mg2+ Tm:58.7˚C 1.5 mM Mg2+ Tm: 66.5˚C 2mM Mg2+ Tm: 67.0˚C
4mM Mg2+ Tm: 68.1˚C 6mM Mg2+ Tm: 68.6˚C
Forward and reverse primer design for insertion into pNIC-Bsa4 (Ruifei): Uh Oh, You have 2 start codons in your forward! - Dr. B
Forward: TACTTCCAATCCATGGAAATCTCTCTCCTC
Reverse: TATCCACCTTTACTGTTATTCTTCGTCCGTCAC
Table 1: GC content and melting temperature as a function of the concentration of Mg++ for CORRECTED** forward and reverse primers for insertion of S. agalactiae serine/threonine phosphatase gene into pNIC-Bsa4 vector with 0.4 uM Oligo concentration, 50 mM Na+ concentration, 0.3 mM dNTP concentration
TMpred graph:
Resources:
GBS Phosphatase: __http://www.jbc.org/content/286/51/44197.long__
Proof that A909 causes sepsis: __http://www.straininfo.net/genomes/326__
PDB: __http://www.rcsb.org/pdb/explore/explore.do?structureId=2PK0__
NCBI: __http://www.ncbi.nlm.nih.gov/protein/AAL58473.1__
Pubmed on neonata sepsis: __http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0004557/__
ExPASy: __http://enzyme.expasy.org/cgi-bin/enzyme/enzyme-search-ec__
SIGMA for PnPP: __http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US__
__http://www.sigmaaldrich.com/technical-documents/articles/biofiles/colorimetric-alkaline.html__
SIGMA FOR ENZYME ASSAY: __http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf__
Price of pNPP on SIGMA: __http://www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=US__
PDB structure of pNPP with phosphotase, etc: __http://www.rcsb.org/pdb/explore/explore.do?structureId=1D1Q__
PDB Structure for STP 1: __http://www.rcsb.org/pdb/explore/images.do?structureId=2PK0__
COMBINED PROTEIN RESULTS: __http://web.expasy.org/cgi-bin/protparam/protparam__
ARticle used for purification: __http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05845.x/full__